DNA adducts represent internal dosimeters to measure exposure to environmental and endogenous genotoxicants. Unfortunately, in epidemiologic studies, measurement of DNA adducts often is precluded by the un- availability of fresh tissue. In contrast, formalin-fixed paraffin embedded (FFPE) tissues with clinical diagnosis are frequently accessible. The screening of DNA adducts in FFPE tissue has been largely limited to immuno- histochemistry (IHC), when antibodies are available. However, an important drawback of IHC is that the specificity of many antibodies, even monoclonal antibodies, for DNA adducts is uncertain as they can cross-react with other DNA lesions, leading to errors in identification and quantification. We recently reported that DNA adducts of aristolochic acid-I (AA-I), can be measured, by mass spectrometric (MS) methods, in human FFPE kidney at a level of sensitivity comparable to that of freshly frozen tissue. AA-I is a carcinogenic compound found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. We propose to adapt our method of DNA retrieval and demonstrate that DNA adducts of other environmental and dietary genotoxicants can be measured in FFPE tissues. The identification of carcinogen DNA adducts in FFPE tissue may provide clues to the origin of human cancers for which an environmental cause is suspected. The objective of this proposal is to show the versatility of our DNA retrieval method through the analysis of DNA adducts of three important classes of human carcinogens: aromatic amines, polycyclic aromatic hydro- carbons (PAHs), and N-nitroso compounds. These chemicals occur in the environment and/or arise in tobacco smoke. The major DNA adducts of these carcinogens are well characterized. Aromatic amines, PAHs and N- nitroso compounds form adducts at different nucleophilic sites of DNA bases and thus, have been selected to evaluate the versatility of our method of DNA retrieval from FFPE tissue. In Aim 1, we will measure DNA adducts in several freshly frozen and FFPE tissues of rodents, following exposure to 4-aminobiphenyl, benzo[a]pyrene, or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. In Aim 2, the DNA retrieval method will be employed to measure these DNA adducts in freshly frozen and FFPE urinary bladder and lung tissues, target organs of cancer in human smokers exposed to these chemicals. An IHC method will be used to screen for 4- ABP-DNA adducts in humans to directly compare current methods to MS. Our proposed research is directly relevant to the Funding Opportunity Announcement issued by the National Cancer Institute. Our technology will advance the usage of a largely untapped biospecimen, FFPE tissue, to measure exposures to carcinogens. Upon validation of the DNA retrieval technology, the employment of FFPE tissue to measure DNA adducts can accelerate our understanding of exposures to genotoxicants in the environment, diet, and tobacco for epidemiologic studies assessing cancer risk and health disparities.